This invention relates to a human platelet analog for use as a reference control in automated particle counting instrumentation of the Coulter type, and by microscope techniques.
Whole blood hematological reference control compositions are now made from stabilized human erythrocytes, preserved human erythrocytes as leukocyte analogs, and stabilized human platelets. Human platelets (thrombocytes) are round or oval disks, about 1/3 to 1/2 the diameter of the erythrocytes (red blood cells) found in human blood. They contain no hemoglobin (red coloring matter) and normally number from 150,000 to 350,000 per mm.sup.3 in normal whole blood.
Previously available reference control products for checking the performance characteristics of particle analysis instruments for determining platelet parameters have suffered from the disadvantage of limited availability or high cost of human blood platelets and the disadvantage of poor stability and/or inconvenient methods of use, especially for automated particle counting instruments. Commercial experience has clearly indicated that the use of human platelets is beset with serious manufacturing cost, and a concern for the use of human blood resources for in vitro diagnostic products which might be contrary to a need to develop a national voluntary blood donor program.
A control product must accurately indicate on a comparative basis what a test sample of blood constitutes in a particular determination. Furthermore, it is evident how important it is for the control product to simulate blood collected in commonly used anticoagulants. For example, if the control product contains cells which are larger in size, the experimental result may be inaccurate in many types of automated equipment, if not almost meaningless.
Any system for automated platelet counting which distinguishes human platelets from other cells in the blood on the basis of the characteristic size range and volume distribution of platelets requires that the reference control material used as such closely simulates the size range and volume distribution characteristics of platelets in normal human blood. A reference control containing platelets or simulated platelets which has a narrow size distribution range would not be useful to determine whether the size distribution limits, between which the instrument counted "platelets", were correctly set. Both the upper and lower size limits of platelets must be represented in the reference control material. In addition, the mean platelet volume of the reference control material should be very close to that of normal human platelets. When upper and lower size limits and mean platelet volume are thus specified, it becomes a virtual necessity for the volume distribution histogram of the platelet material to approximate closely the log-normal distribution of fresh human platelets.
A comparison of the volume distribution histogram of the platelets in fresh, human whole blood with the volume distribution histogram of a typical commercial platelet reference control made from human platelets shows that the modal point of the distribution of the commercial reference control platelet suspension is significantly lower than that of the platelets in fresh blood. In addition, the low-volume end of the histogram for the reference control material is lower than that found for the fresh platelets. This would seem to indicate that the preservation process now used in the manufacture of the reference control suspension caused significant shrinkage of platelets.
Other commercial platelet reference control preparations suffer on aging from deterioration of the volume distribution histogram characteristics as well as deterioration of other parameters. Thus, the usefulness of a given lot number of a platelet reference control or a whole blood control containing platelets can be limited by the lack of stability of the assigned value.
There appears to be an inherent incompatibility between the need to stabilize the reference control platelets for the purpose of obtaining good product shelf life and the maintenance of the size range, mean volume and log-normal size distribution histogram which are characteristic of normal platelets. The solution to the problem lies not in the pursuit of more effective ways of stabilizing "real" (human) platelets, but in substituting a surrogate which satisfies the specifications against which the product is made. Animal platelets are not useful because they are small in number, and also tend to clump together.
With the increasing use of automated devices capable of performing multiple hematological determinations and with the introduction of techniques of automated cell counting, there has been an increasing need for the development of particles which can be used either for reference control purposes or as calibrators.
Three principal types of particles heretofore have been investigated namely, human or animal cells, non-animal cells such as yeasts or pollens, and synthetic particles such as polystyrene latex. Latex particles, while capable of being manufactured to very close mean volume and size distribution specifications, present serious problems in achieving smooth, uniform suspensions. Pollens and yeasts, in addition to sharing the suspension stability problems of latex particles, suffer from lack of uniformity from batch to batch, and in some cases, lack of availability. A further requirement for the platelet component in a whole blood reference control for multi-parameter instruments is that the cells must be lysed by the lytic reagent. Latex particles and non-animal cells lack this property.